Intracoronary dual-modal optical coherence tomography-near-infrared fluorescence structural-molecular imaging with a clinical dose of indocyanine green for the assessment of high-risk plaques and stent-associated inflammation in a beating coronary artery.

AIMS: Inflammation plays essential role in development of plaque disruption and coronary stent-associated complications. This study aimed to examine whether intracoronary dual-modal optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural-molecular imaging with indocyanine green (ICG) can estimate inflammation in swine coronary artery. METHODS AND RESULTS: After administration of clinically approved NIRF-enhancing ICG (2.0 mg/kg) or saline, rapid coronary imaging (20 mm/s pullback speed) using a fully integrated OCT-NIRF catheter was safely performed in 12 atheromatous Yucatan minipigs and in 7 drug-eluting stent (DES)-implanted Yorkshire pigs. Stronger NIRF activity was identified in OCT-proven high-risk plaque compared to normal or saline-injected controls (P = 0.0016), which was validated on ex vivo fluorescence reflectance imaging. In vivo plaque target-to-background ratio (pTBR) was much higher in inflamed lipid-rich plaque compared to fibrous plaque (P < 0.0001). In vivo and ex vivo peak pTBRs correlated significantly (P < 0.0022). In vitro cellular ICG uptake and histological validations corroborated the OCT-NIRF findings in vivo. Indocyanine green colocalization with macrophages and lipids of human plaques was confirmed with autopsy atheroma specimens. Two weeks after DES deployment, OCT-NIRF imaging detected strong NIRF signals along stent struts, which was significantly higher than baseline (P = 0.0156). Histologically, NIRF signals in peri-strut tissue co-localized well with macrophages. CONCLUSION: The OCT-NIRF imaging with a clinical dose of ICG was feasible to accurately assess plaque inflammation and DES-related inflammation in a beating coronary artery. This highly translatable dual-modal molecular-structural imaging strategy could be relevant for clinical intracoronary estimation of high-risk plaques and DES biology.

High-speed 3-D measurement with a large field of view based on direct-view confocal microscope with an electrically tunable lens.

We propose a new structure of confocal imaging system based on a direct-view confocal microscope (DVCM) with an electrically tunable lens (ETL). Since it has no mechanical moving parts to scan both the lateral (x-y) and axial (z) directions, the DVCM with an ETL allows for high-speed 3-dimensional (3-D) imaging. Axial response and signal intensity of the DVCM were analyzed theoretically according to the pinhole characteristics. The system was designed to have an isotropic spatial resolution of 20 µm in both lateral and axial direction with a large field of view (FOV) of 10 × 10 mm. The FOV was maintained according to the various focal shifts as a result of an integrated design of an objective lens with the ETL. The developed system was calibrated to have linear focal shift over a range of 9 mm with an applied current to the ETL. The system performance of 3-D volume imaging was demonstrated using standard height specimens and a dental plaster.

High-speed time-resolved laser-scanning microscopy using the line-to-pixel referencing method.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to visualize photophysical characteristics of biological targets. However, conventional FLIM methods have some limitations that restrict obtaining high-precision images in real time. Here, we propose a high-speed time-resolved laser-scanning microscopy by incorporating a novel line-to-pixel referencing method into the previously suggested analog mean-delay (AMD) method. The AMD method dramatically enhances the photon accumulation speed for achieving the certain precision compared to the time-correlated single-photon counting (TCSPC) method while maintaining high photon efficiency. However, its imaging pixel rate can still be restricted by the rearm time of the digitizer when it is triggered by laser pulses. With our line-to-pixel referencing method, the pulse train repeats faster than the trigger rearm time can be utilized by generating a line trigger, which is phase-locked with only the first pulse in each horizontal line composing an image. Our proposed method has been tested with a pulsed laser with 40 MHz repetition rate and a commercial digitizer with a 500 ns trigger rearm time, and a frame rate of 3.73 fps with a pixel rate of 3.91 MHz was accomplished while maintaining the measurement precision under 20 ps.

Chromatic confocal microscopy with a novel wavelength detection method using transmittance.

Chromatic confocal microscopy (CCM) is a promising technology that enables high-speed three-dimensional surface profiling without mechanical depth scanning. However, the spectrometer, which measures depth information encoded by axial color, limits the speed of three-dimensional imaging. We present a novel method for chromatic confocal microscopy with transmittance detection. Depth information can be instantaneously obtained by the ratio of intensity signals from two photomultiplier tubes by detecting a peak wavelength using transmittance of a color filter. This non-destructive and high-speed surface profiling method might be useful in many fields, including the semiconductor and flat panel display industries, and in material science.

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

Polarization-sensitive spectral-domain optical coherence tomography using a multi-line single camera spectrometer.

We describe a polarization sensitive spectral domain optical coherence tomography technique based on a single camera spectrometer that includes a multiplexed custom grating, camera lenses, and a high-speed three-line CCD camera. Two orthogonally polarized beams could be separately taken by two lines of the camera as a result of vertically different incident angles. The system could provide the imaging capabilities of a full camera speed and increased measurable depth. The proposed optical coherence tomography system could make a distinction between the normal muscle and cancerous tissue from the chest of a DSred GFP mouse and the OCT images were compared with those of in vivo confocal microscopy.

Development of compact high precision two degree of freedom XY piezoelectric stepping positioner.

In this article, a linear piezoelectric stepping positioner developed by Kang et al. [Rev. Sci. Instrum. 78, 075112 (2007)] is extended to have a two degree of freedom XY linear motion and it is experimentally evaluated. A resolution less than 10 nm, a speed of over 0.5 mm/s, push forces of 17.9 and 19.5, and stiffnesses of 6.54 and 5.90 N/microm are attained while maintaining a compact size of 96x96x39 mm3 and the required power consumption of 102.74 W.

Design and control of a nanoprecision XY Theta scanner.

This paper describes the design and control of a nanoprecision XY Theta scanner consisting of voice coil motors and air bearing guides. The proposed scanner can be installed on a conventional XY stage with long strokes to improve the positioning accuracy and settling performance. Major design considerations in developing a high precision scanner are sensor accuracy, actuator properties, structural stability, guide friction, and thermal expansion. Considering these factors, the proposed scanner is made of invar, which has a small thermal expansion coefficient and good structural stiffness. Four voice coil motors drive the scanner, which is suspended by four air bearing pads, in the x, y, and theta directions. The scanner's position is measured by three laser interferometers which decouple the scanner from the conventional stage. The mirror blocks reflecting the laser beams are fixed using viscoelastic sheets, ensuring that the scanner has a well-damped structural mode. A time delay control algorithm is implemented on the real-time controller to control the scanner. The effectiveness of the proposed scanner is verified experimentally.

Therapeutic Effects of Targeted PPARÉ£ Activation on Inflamed High-Risk Plaques Assessed by Serial Optical Imaging In Vivo.

Rationale: Atherosclerotic plaque is a chronic inflammatory disorder involving lipid accumulation within arterial walls. In particular, macrophages mediate plaque progression and rupture. While PPARγ agonist is known to have favorable pleiotropic effects on atherogenesis, its clinical application has been very limited due to undesirable systemic effects. We hypothesized that the specific delivery of a PPARγ agonist to inflamed plaques could reduce plaque burden and inflammation without systemic adverse effects. Methods: Herein, we newly developed a macrophage mannose receptor (MMR)-targeted biocompatible nanocarrier loaded with lobeglitazone (MMR-Lobe), which is able to specifically activate PPARγ pathways within inflamed high-risk plaques, and investigated its anti-atherogenic and anti-inflammatory effects both in in vitro and in vivo experiments. Results: MMR-Lobe had a high affinity to macrophage foam cells, and it could efficiently promote cholesterol efflux via LXRα-, ABCA1, and ABCG1 dependent pathways, and inhibit plaque protease expression. Using in vivo serial optical imaging of carotid artery, MMR-Lobe markedly reduced both plaque burden and inflammation in atherogenic mice without undesirable systemic effects. Comprehensive analysis of en face aorta by ex vivo imaging and immunostaining well corroborated the in vivo findings. Conclusion: MMR-Lobe was able to activate PPARγ pathways within high-risk plaques and effectively reduce both plaque burden and inflammation. This novel targetable PPARγ activation in macrophages could be a promising therapeutic strategy for high-risk plaques.

Development of compact high precision linear piezoelectric stepping positioner with nanometer accuracy and large travel range.

Many application areas such as semiconductor manufacture, precision optics alignment, and microbiological cell manipulation require ultraprecision positioning systems with a high positioning resolution and large motion range. This article describes the development of a compact high precision linear piezoelectric stepping positioner for precision alignment of optical elements. The positioner is designed to have a compact and symmetric structure, high positioning resolution, large motion range, high force density, adequate dynamic range, and power-off hold. The positioner is fabricated according to these specifications and performance evaluation tests are carried out. A resolution of 10 nm, speed of 1 mms, push force of 25 N, and stiffness of 10.4 N/microm are attained while maintaining a compact size of 32x42x60 mm(3). The required power consumption is 52.33 W. The test results confirm that the developed positioner could be successfully applied to the precision alignment of optical elements.

Processable high internal phase Pickering emulsions using depletion attraction.

High internal phase emulsions have been widely used as templates for various porous materials, but special strategies are required to form, in particular, particle-covered ones that have been more difficult to obtain. Here, we report a versatile strategy to produce a stable high internal phase Pickering emulsion by exploiting a depletion interaction between an emulsion droplet and a particle using water-soluble polymers as a depletant. This attractive interaction facilitating the adsorption of particles onto the droplet interface and simultaneously suppressing desorption once adsorbed. This technique can be universally applied to nearly any kind of particle to stabilize an interface with the help of various non- or weakly adsorbing polymers as a depletant, which can be solidified to provide porous materials for many applications.

Quantitative analysis of methyl parathion pesticides in a polydimethylsiloxane microfluidic channel using confocal surface-enhanced Raman spectroscopy.

A fast and ultra-sensitive trace analysis of methyl parathion pesticides in a polydimethylsiloxane (PDMS) microfluidic channel was investigated using confocal surface-enhanced Raman spectroscopy (SERS). A three-dimensional PDMS-based passive micromixer was fabricated for this purpose. This PDMS micromixer showed a high mixing efficiency because a strong chaotic advection was developed by the simultaneous vertical and transverse dispersion of the confluent streams. The confocal SERS signal was measured after methyl parathion pesticides were effectively adsorbed onto silver nanoparticles while flowing along the upper and lower alligator-teeth-shaped PDMS channel. A quantitative analysis of the methyl parathion pesticides was performed based on the measured peak height at 1246 cm-1. Our method has a detection limit of 0.1 ppm. This value satisfies the requirement recommended by the Collaborative International Pesticides Analytical Council (CIPAC) for the determination of methyl parathion in pesticide formulations. This study demonstrates the feasibility of using confocal SERS for the highly sensitive detection of methyl parathion pesticides in a PDMS microfluidic channel.

Note: Development of a compact aperture-type XYθz positioning stage.

In this paper, we propose a new in-plane XYθz nano-positioning stage that utilizes piezoelectric actuators and flexure mechanisms. The proposed stage has an aperture and is compact, which facilitates its application in measurement equipment, especially those used for biological specimens. The stage has four piezoelectric actuators and four bridge-type flexure mechanisms, which are used to amplify the small motions produced by the piezoelectric actuators. This paper describes the modeling and design optimization of the stage, which has X- and Y-direction motion ranges of 300 µm and a θz-direction motion range of ±3.9 mrad. The stage measures 150 × 150 × 23 mm, and its aperture is 50 × 50 mm.

Intravascular optical imaging of high-risk plaques in vivo by targeting macrophage mannose receptors.

Macrophages mediate atheroma expansion and disruption, and denote high-risk arterial plaques. Therefore, they are substantially gaining importance as a diagnostic imaging target for the detection of rupture-prone plaques. Here, we developed an injectable near-infrared fluorescence (NIRF) probe by chemically conjugating thiolated glycol chitosan with cholesteryl chloroformate, NIRF dye (cyanine 5.5 or 7), and maleimide-polyethylene glycol-mannose as mannose receptor binding ligands to specifically target a subset of macrophages abundant in high-risk plaques. This probe showed high affinity to mannose receptors, low toxicity, and allowed the direct visualization of plaque macrophages in murine carotid atheroma. After the scale-up of the MMR-NIRF probe, the administration of the probe facilitated in vivo intravascular imaging of plaque inflammation in coronary-sized vessels of atheromatous rabbits using a custom-built dual-modal optical coherence tomography (OCT)-NIRF catheter-based imaging system. This novel imaging approach represents a potential imaging strategy enabling the identification of high-risk plaques in vivo and holds promise for future clinical implications.

Highly sensitive signal detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip: confocal surface-enhanced Raman spectroscopic study.

Rapid and highly sensitive detection of duplex dye-labelled DNA sequences in a PDMS microfluidic channel was investigated using confocal surface enhanced Raman spectroscopy (SERS). This method does not need either an immobilization procedure or a PCR amplification procedure, which are essential for a DNA microarray chip. Furthermore, Raman peaks of each dye-labelled DNA can be easily resolved since they are much narrower than the corresponding broad fluorescence bands. To find the potential applicability of confocal SERS for sensitive bio-detection in a microfluidic channel, the mixture of two different dye-labelled (TAMRA and Cy3) sex determining Y genes, SRY and SPGY1, was adsorbed on silver colloids in the alligator teeth-shaped PDMS microfluidic channel and its SERS signals were measured under flowing conditions. Its major SERS peaks were observable down to the concentration of 10(-11) M. In the present study, we explore the feasibility of confocal SERS for the highly sensitive detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip.

Fiber-optic raster scanning two-photon endomicroscope using a tubular piezoelectric actuator.

A nonresonant, fiber-optic raster scanning endomicroscope was developed using a quarter-tubular piezoelectric (PZT) actuator. A fiber lever mechanism was utilized to enhance the small actuation range of the tubular PZT actuator and to increase its field-of-view. Finite element method simulation of the endoscopic probe was conducted for various conditions to maximize its scanning range. After fabricating the probe using a double clad fiber, we obtained two-photon fluorescence images using raster beam scanning of the fiber. The outer diameter of the probe was 3.5 mm and its rigid distal length was 30 mm including a high numerical aperture gradient index lens. These features are sufficient for input into the instrumental channel of a commercial colonoscope or gastroscope to obtain high resolution images in vivo.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging.

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Optimal design and experiment of a three-axis out-of-plane nano positioning stage using a new compact bridge-type displacement amplifier.

This paper presents the development of a new compact three-axis compliant stage employing piezoelectric actuators and a new flexure structure. A proposed stage works out-of-plane (Z, θx, θy) direction. The stage consists of 4 amplification flexures mounted piezoelectric actuators. New structure of flexure reduces height and enhances dynamic performance of stage. To certify excellent performance of the stage, comparison accomplished between conventional amplification flexure and new compact bridge type flexure. Modeling and optimal design of new type nano positioning stage performed. The optimal design is executed on the geometric parameters of the proposed flexure structure using Sequential Quadratic Programming. Experiments are carried out to verify the static and dynamic performance of the stage. The proposed out-of-plane nano-positioning stage has a Z-directional motion range 190 µm and a θx, θy-directional motion range ±2 mrad. The resolution of the stage is 4 nm, 40 nrad, and 40 nrad in the Z-, θx-, and θy-directional motions, respectively. The size of stage is 150 × 150 × 30 mm(3).

Development of a compact and long range XYθ(z) nano-positioning stage.

In this study, we describe the development of a novel, compact, and long range in-plane XYθ(z) nano-positioning stage with piezoelectric actuator and flexure mechanism. The stage is composed of an X-directional motion part and a Y, θ(z)-directional motion part, which are linked serially. The stage consists of a bridge-type amplifying mechanism for the amplification of deformation of the piezoelectric actuator, a double compound guide mechanism for performing only desired motion, and a circular hinge mechanism that permits rotational motion in the Y and θ(z)-stages. To set the design variables of the stage, optimal design is carried out. To verify the results of the optimal design process and the performance of the stage, the FEM simulation and experiment are carried out. The proposed XYθ(z) nano-positioning stage has a translational motion range of 700 µm and a rotational motion range of 0.3°; it has a closed-loop resolution of 5 nm, 5 nm, and 0.025 arcsec in the X-, Y-, and θ(z)-directional motions, respectively. The proposed stage is a novelty in that it has a compact size of 200 × 200 × 30 mm(3), and decoupled kinematic design.

Development of flexure based 6-degrees of freedom parallel nano-positioning system with large displacement.

This paper details the development of a novel flexure jointed precision parallel nano-positioning system in combination with piezo-electric stepping motor for the application of precise optics alignment. The characteristics of the developed system are evaluated in this paper by the simulation and experiments. Based on the precision piezo-electric stepping motor and flexure joints, a high precision motion is obtained. Results of this paper include that of a translation resolution of 15 nm and a rotational resolution of 0.14 arc sec being achieved. In addition, the piezo-electric stepping motors provide a power-off hold characteristic to the system. Meanwhile, the parallel structure provides the high dynamic bandwidth of the lowest resonant frequency of 396.1 Hz. The symmetric structure is advantageous for thermal variation. To increase the motion range of the system, all of flexure joints are designed specially and the coupled workspace of ±2 mm × ±2 mm × ±2 mm × ±2° × ±2° × ±2° is achieved. The overall size of the designed system is Φ350 mm × 120 mm.